Hannover, Universiẗat, Diss., 2004.

This work aimed to investigate the evolutionary origin of the involvement of the HIF Prolyl Hydroxylases (PHDs) in oxygen sensing. The &alpha;/&beta;-heterodimer HIF (<u>H</u>ypoxia <u>I</u>nducible <u>F</u>actor) is a master regulator of oxygen homoeostasis in metazoans. In the nucleus, HIF binds to the Hypoxia Responsive Elements and forms a transcriptional complex that activates the transcription of a multitude of downstream genes. Under normoxic conditions, the Fe(II)- and oxygen-dependent PHDs catalyse 4R-prolyl-hydroxylation of the HIF &alpha;-subunit, which subsequently leads to its degradation. It had previously been proposed that the evolution of the HIF-pathway, shared by all metazoans but not found in other organisms, is linked to the rapid diversification of multicellular life during the Cambrian Explosion. This work investigates the structural and biochemical properties of a PHD of the basal metazoan Trichoplax adhaerens (taPHD), and a PHD-like enzyme of the protist Monosiga brevicollis (mbP4H). Two crystal structures of taPHD were obtained (1.2-1.3 Å), one containing a Trichoplax adhaerens HIF&alpha; subunit peptide (taODD). Comparison with crystal structures of human PHD2 showed a high degree of conservation of structural features and enzyme-substrate interactions. The prolyl-residue of taODD, shown to be hydroxylated by taPHD, is occupying the C⁴-endo conformation in the crystal structure, supporting the previously proposed mechanism of HIF&alpha; hydroxylation by PHD2 in humans. A conservation of biochemical properties with human PHD2, such as the formation of a stable enzyme-Fe(II)-2OG complex, was observed and could therefore be key to oxygen sensing by the PHDs. mbP4H was shown to catalyse 4R-prolyl-hydroxylation of taODD. It was proposed that the native substrate of mbP4H is a protein containing a prolyl-hydroxylation site similar to taODD, possibly with a YXXLAP motif. The study of biochemical properties and substrate selectivity of mbP4H suggests that the precursor of PHDs may have had similar properties to mbP4H. Further work on mbP4H could therefore yield clues about the evolutionary origin of HIF-prolyl hydroxylases in oxygen sensing and probe the previously proposed connection between metazoan life and HIF–mediated oxygen sensing.

Der internal transcribed spacer 2 (ITS2) des ribosomalen Genrepeats ist ein zunehmend wichtiger phylogenetischer Marker, dessen RNA-Sekundärstruktur innerhalb vieler eukaryontischer Organismen konserviert ist. Die ITS2-Datenbank hat zum Ziel, eine umfangreiche Ressource für ITS2-Sequenzen und -Sekundärstrukturen auf Basis direkter thermodynamischer als auch homologiemodellierter RNA-Faltung zu sein. Ergebnisse: (a) Eine komplette Neufassung der ursprünglichen die ITS2-Datenbank generierenden Skripte, angewandt auf einen aktuellen NCBI-Datensatz, deckte mehr als 65.000 ITS2-Strukturen auf. Dies verdoppelt den Inhalt der ursprünglichen Datenbank und verdreifacht ihn, wenn partielle Strukturen mit einbezogen werden. (b) Die Endbenutzer-Schnittstelle wurde neu geschrieben, erweitert und ist jetzt in der Lage, benutzerdefinierte Homologiemodellierungen durchzuführen. (c) Andere möglichen RNA-Strukturaufklärungsmethoden (suboptimales und formenbasiertes Falten) sind hilfreich, können aber Homologiemodellierung nicht ersetzen. (d) Ein Anwendungsfall der ITS2-Datenbank in Zusammenhang mit anderen am Lehrstuhl entwickelten Werkzeugen gab Einblick in die Verwendung von ITS2 für molekulare Phylogenie. / The internal transcribed spacer 2 (ITS2) of the ribosomal gene repeat is an increasingly important phylogenetic marker whose RNA secondary structure is widely conserved across eukaryotic organisms. The ITS2 database aims to be a comprehensive resource on ITS2 sequence and secondary structure, based on direct thermodynamic as well as homology modelled RNA folds. Results: (a) A rebuild of the original ITS2 database generation scripts applied to a current NCBI dataset reveal more than 60,000 ITS2 structures. This more than doubles the contents of the original database and triples it when including partial structures. (b) The end-user interface was rewritten, extended and now features user-defined homology modelling. (c) Other possible RNA structure discovery methods (namely suboptimal and shape folding) prove helpful but are not able to replace homology modelling. (d) A use case of the ITS2 database in conjunction with other tools developed at the department gave insight into molecular phylogenetic analysis with ITS2.

A detailed annotation of non-protein coding RNAs is typically missing in initial releases of newly sequenced genomes. Here we report on a comprehensive ncRNA annotation of the genome of Trichoplax adhaerens, the presumably most basal metazoan whose genome has been published to-date. Since blast identified only a small fraction of the best-conserved ncRNAs—in particular rRNAs, tRNAs and some snRNAs—we developed a semi-global dynamic programming tool, GotohScan, to increase the sensitivity of the homology search. It successfully identified the full complement of major and minor spliceosomal snRNAs, the genes for RNase P and MRP RNAs, the SRP RNA, as well as several small nucleolar RNAs. We did not find any microRNA candidates homologous to known eumetazoan sequences. Interestingly, most ncRNAs, including the pol-III transcripts, appear as single-copy genes or with very small copy numbers in the Trichoplax genome.

Transcriptional regulation of gene expression in eukaryotes has evolved over millions of years. The regulatory pathways of nuclear receptors represent an evolutionarily ancient, but conserved mechanism with associated accessory proteins, many of them forming a functional nexus known as the Mediator complex involved in transcription. Despite the versatility of the pathway, e.g. through the adoption of new regulatory functions in phylogenetically more recent Metazoa, we hypothesise that the intrinsic potential of the NR-Mediator axis to directly translate a stimulus to a biological response is conserved across species, and additional regulation could also be achieved through secondary functions of its essential members. To support the hypothesis, we assessed the ligand-binding capability of retinoic X receptor in Trichoplax adhaerens and provided evidence to support the concept that this capability was already present at the base of metazoan evolution. With regards to the potential secondary functions, we took inspiration from previous research and identified the Mediator subunit 28 (MED28) as the only known member having documented nuclear and cytoplasmic dual roles, and thus possessing the potential to transmit signals from the cellular structural states to the nucleus. Due to the lack of...

In animals, some of the most critical regulators of gene expression are nuclear hormone receptors (NRs) and their coregulators, specifically the Mediator complex. Of particular interest are the NRs implicated in metabolic and developmental regulation and in carcinogenesis: thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). In this work, I venture to elucidate some aspects of gene expression regulation by these NRs: the degree of evolutionary conservation of signalling based on NRs and their coregulators; the mechanisms of negative regulation by NRs; and possible implications of these findings for clinical medicine. State-of-the-art bioinformatical, genome editing and microscopic techniques are applied at three levels of animal evolution to study NRs and Mediator. Reverse genomics in human patients suffering from the syndrome of resistance to thyroid hormones β are used to infer the structure and function of TRβ subdomains. Alignments, binding studies and in vivo experiments in Trichoplax adhaerens allow identification of a close orthologue of human RXR at the basis of metazoan evolution. Employing database queries, genome editing and microscopy, we describe a correct orthologue of the Mediator subunit 28 in Caenorhabditis elegans, indicating a complete homology of the Mediator complex...